CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Effects of supernatant of different hepatoma cells on the vitality of Echinococcus granulosus protoscoleces in vitro
Received date: 2019-11-27
Online published: 2020-07-07
Supported by
National Natural Science Foundation of China(81760570);National Natural Science Foundation of China(81760371);Program for Young and Middle-aged Scientific and Technological Innovation Leaders of the XPCC(2018CB017);Science and Technology Public Relations Projects in Key Areas of the XPCC(2019AB031)
Objective To explore a new model for long-time retainment of the vitality of Echinococcus granulosus protoscoleces in vitro by comparing the effects of supernatant of three hepatocarcinoma cell lines on the vitality of E. granulosus protoscoleces in vitro. Methods Serum-free supernatants of three liver carcinoma cell lines, Huh7, Hepa1-6, and HepG-2, were collected after 48-h culture, and co-cultured with the protoscoleces of E. granulosus (about 5 000 protoscoleces in each group). Protoscoleces of the control group were cultured with serum-free medium only. On days 1, 3, 5 and 7 of culture, 50 μl of the suspension was taken from each group for trypan blue staining to count dead cells by light microscopy, and the survival rate was calculated. On day 7 after culture, the ultrastructure of protoscoleces was observed under a scanning electron microscope, and ELISA was performed to determine the activity of caspase-3 of the four groups. SPSS 20.0 software was used for variance analysis on the measured data. Results Trypan blue staining showed that the survival rate of the protoscoleces continuously decreased with prolonged culture in each group. On day 5 of culture, the survival rate of protoscoleces in the serum-free group, Huh7 group, Hepa1-6 group, and HepG-2 group decreased to (46.54 ± 1.11)%, (46.19 ± 3.60)%, (55.90 ± 2.33)%, and (68.73 ± 1.06)%, respectively. On day 7, the survival rate of protoscoleces in the Hepa1-6 and HepG2 group was (36.09 ± 0.95)% and (45.69 ± 0.40)%, respectively, which were significantly lower than that from control group [(17.99 ± 1.52)%, P < 0.01], while that of the Huh7 group [(19.04 ± 1.70)%] was not significantly different from the control group (P > 0.05). On day 7 of culture, light microscopy indicated more blue-stained protoscoleces with poor light transmittance and more disintegrated and necrotic debris in the control and Huh7 groups; higher numbers of stained protoscoleces but moderate light transmittance and less necrotic debris in the Hepa1-6 group; and small number of stained protoscoleces, high light transmittance and few necrotic debris in the HepG-2 group. Scanning electron microscopy showed that the morphology of the protoscoleces of the control group and Huh7 group was glargely destroyed, and the ultrastructures of microhairs and acetabula were disrupted and disintegrated. The protoscoleces of the Hepa1-6 and HepG2 groups showed a plump and smooth surface as well as clear and intact ultrastructure. The enzyme activity unit of Caspase-3 of the Hepa1-6 group and HepG2 group was (20.51 ± 0.61) and (17.51 ± 0.59) on day 7, which were statistically different from the control group (22.15 ± 1.14) (P < 0.05, 0.01). There was no statistically significant difference in enzymatic activity between the Huh7 group (22.67 ± 0.96) and the control group (P > 0.05). Conclusion The supernatant of hepatocarcinoma cell culture can improve the vitality of E. granulosus protoscoleces under serum-free culture in vitro, and particularly, culture presents more obvious effect.
He-jie CHEN , Hui-jiao JIANG , Qian LIANG , Jie Wu , Xian-wei GUI , Hai-liang ZOU , Zhi-kun XING , Er-qiang WANG , Xue-ling CHEN , Xiang-wei WU . Effects of supernatant of different hepatoma cells on the vitality of Echinococcus granulosus protoscoleces in vitro[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(3) : 317 -323 . DOI: 10.12140/j.issn.1000-7423.2020.03.010
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